The present invention relates to a novel pharmaceutical or food composition intended for treating pathologies associated with graft rejection or an allergic or autoimmune reaction.
In the last twelve years, controlled studies have described desensitization based on the oral administration of allergens (1). This method is based on the fact that the oral administration of an antigen facilitates the acquisition of an immunological tolerance to it. The digestive route constitutes the mode of contact of the body with antigens, of food or microbial origin. However, allergic reactions are rare. Oral administration of sheep red blood cells (SRBC) to rats prevents the rats from later producing anti-SRBC antibodies after a subcutaneous injection, whereas, without the prior oral intake, the allergic response would have been present. This phenomenon constitutes what is referred to immunologically as oral tolerance.
This oral desensitization method has been validated in prospective and controlled studies, and makes it possible to reduce the risks of anaphylaxis, in particular for birch pollen and acari. It is already available on the vaccines market in a presentation in drinkable form (sold by the company Laboratoire des Stallergxc3xa9nesxe2x80x94Paris).
Moreover, it may be considered that the benefit, in terms of protecting infants against allergy to milk, which has been observed since the introduction of new, enzymatically predigested powdered milk formulations, would result from the induction of immunological tolerances by the presentation of antigens in the form of peptides.
Document WO-A-95/24920 describes the use of complexes consisting of the combination of a stress protein and a peptide as a prophylactic or therapeutic vaccine against intracellular pathogens.
That document does not in any way describe the possibility of using such complexes in vivo in the treatment of autoimmune or allergic diseases or graft rejections.
In the third paragraph of page 10, that document describes that said immunogenic and antigenic macromolecular structures can be associated with autoimmune or allergic diseases, and that, in this case, said antigen is administered in combination with said stress proteins in an amount which is sufficient to generate tolerance or to inhibit a pre-existing immune response against said antigen in an individual.
It is indicated that the amount of stress proteins required to inhibit this immune response is assumed to be substantially larger than the amounts required to obtain a stimulation.
However, it is difficult to predict or state the efficacy of the desensitization. Clinical observation makes it possible, after the event, to confirm or deny any improvement in the symptoms.
It is known, from international patent application WO 96/36880, to be able to detect and/or quantify ligands specific to a pathology associated with an allergic or autoimmune response or lung cancer, by means of a test of competition between ligands present in a sample and other discriminable ligands. This test is based on the fact that allergic and symptomatic individuals recognize, by means of their antibodies, epitopes different from those recognized by the antibodies of tolerant individuals on the same specific immunogenic and antigenic macromolecular structure of said pathology. That document also describes the possibility of measuring the evolution of this specificity, in particular in the case of children who are allergic to milk and the change toward the in vivo acquisition of tolerance to milk.
Patent application WO94/29459, xe2x80x9cStress proteins and uses thereofxe2x80x9d relates to a stress protein and methods of modulating an xe2x80x9cindividualxe2x80x9d immune response. It also relates to a composition comprising a stress protein joined to another component, such as a fusion protein in which a stress protein is fuse to an antigen. According to the invention, it is possible to modulate the immune response in an individual, such as a human, other mammals or other vertebrates by altering the individual""s response to stress protein. By altering the individual""s immune response to the stress protein, it is possible to enhance or induce an individual""s response to a pathogen or to cancer cells or enhance or induce an up-regulation of an individual""s immune status and to decrease an individual""s autoimmune response, such as it occurs in some forms of arthritis.
U.S. Pat. No. 6,048,530 xe2x80x9cStress protein-peptide complexes as prophylactic and therapeutic vaccines against intracellular pathogensxe2x80x9d describes a family of vaccines that contain stress protein-peptide complexes which, when they are administered to a mammal, are operative to initiate in the mammal a cytotoxic T cell response against cells infected with a pre-selected intracellular pathogen. The patent also discloses the preparation of stress protein and immunogenic stress protein-peptide complexes from infected cells or tissues, the isolation of potentially immunogenic peptides from stress protein-peptide complexes from infected cells or from MHC-peptides complexes. The synthesis of those peptides and the reconstitution of heat shock protein-peptide complexes with synthetic peptides or peptides isolated from heat shock protein-peptide complexes isolated from infected cells is also described. According to said document, the peptides-heat shock protein complexes, when isolated from a eukaryotic cell infected with a pre-selected intracellular pathogen, and then administered to a mammal can stimulate a cytotoxic T cell response against cells infected with the same pathogen.
Patent application WO97/06821 xe2x80x9cHeat shock protein-based vaccines and immunotherapiesxe2x80x9d describes a composition for inducing a therapeutic immune response in a subject, comprising: a) a target antigen; and b) a heat shock protein; wherein the target antigen and the heat shock protein are combined in vitro under conditions wherein binding of target antigen to the heat shock protein occurs to form a target antigen-heat shock protein complex; wherein the administration of the target antigen/heat shock protein complex to the subject induces an immune response comprising a cytotoxic cellular component.
Patent application WO98/23735 xe2x80x9cImmune responses using compositions containing stress proteinsxe2x80x9d describes a vaccine for inducing a cell-mediated cytolytic immune response in a mammal against an antigen. The vaccine is comprising the antigen and all or a portion of stress protein or all or a portion of a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of the stress protein to induce the immune response against the antigen. In one embodiment, the antigen is an antigen of the influenza virus. In another embodiment, the antigen is a tumor-associated antigen. The patent also describes a mixture of known allergenic antigens (allergens) and stress proteins or compositions containing allergens chemically linked or fused to the stress protein. Allergens used in allergen-stress protein fusion proteins are necessarily of a peptidic nature; non peptidic allergens can be used in conjugates containing an allergen and a stress protein or a mixture of allergens and stress proteins. Non limiting examples for allergens include Fel d1 (cat), . . .
Epitope mapping technology is the identification and localization of the specific regions of macromolecules that are recognized by the immune system. An epitope is a small region of a macro molecule that the immune system recognizes and responds to. T cell epitopes are linear fragments of the original protein molecule whereas B cell epitopes can be either linear fragments or folded three-dimensional regions of the intact macro molecule.
Several methods for epitopes mapping have been previously described:
Defined fragments of the cDNA for the antigens of interest are expressed as recombinant (fusion)proteins and probed with autoantisera in various assays such as Western blot or ELISA.
Small random fragments of the cDNA for the antigen of interest are cloned into the phage protein pIII or pVIII of the filamentous phage and are displayed on the surface of the phage. Epitope-displaying phages can be captured with antibodies in a procedure called xe2x80x9cbio-panningxe2x80x9d. Sequencing of the inserts of the corresponding phages gives some information on the epitopes. This procedure is in principle capable to identify conformational epitopes.
Small overlapping oligopeptides that ideally cover the complete amino acid sequence of the antigen of interest are synthesized on a solid support and probed with antisera. This method allows the identification of linear epitopes on the amino acid level. It also allows rapid mutational studies.
Prior art provides neither a method to prepare in-vitro a mixture between at least one heat shock protein and peptides resulting from the in-vitro hydrolysis of at least one immunogenic protein, nor a method to identify from such a mixture the peptides that can bind to the stress proteins and/or the peptides that do not bind to the stress protein.
Prior art describes neither compositions according to the invention comprising a mixture of at least one heat shock proteins and peptides resulting from the in-vitro hydrolysis of at least one immunogenic protein and antigenic macromolecular structure, nor a method to prepare such a composition.
Prior art describes neither compositions according to the invention comprising a mixture of heat shock proteins, complexes between the heat shock proteins and the peptides resulting from said in-vitro hydrolysis, and free peptides resulting from the in-vitro hydrolysis nor a method to prepare such a composition.
Prior art describes neither a composition according to the invention comprising purified complexes between heat shock proteins and some of the peptides resulting from said in-vitro hydrolysis nor a method to prepare such a composition.
Prior art describes neither compositions according to the invention comprising peptides that do not bind to heat shock proteins, these peptides resulting from the said in-vitro hydrolysis nor a method to prepare such a composition.
Prior art describes neither compositions comprising the peptides isolated from the complexes made between Heat Stock Proteins (HSP) and peptides resulting from said in-vitro hydrolysis nor a method to prepare such a composition.
The aim of the present invention is to provide a novel composition, which may be of pharmaceutical or food type, designed to modify the immune response of patients toward a pathology associated with an allergic or autoimmune reaction or toward graft rejection phenomena, such that the immune response of said patients comes close to the natural tolerance manifested by normal individuals (who remain free of symptoms although they are also liable to be exposed to this pathology).
The present invention is also directed toward providing an inexpensive pharmaceutical or food composition which is easy to administer and which can be used in a prophylactic and/or therapeutic manner.
A further aim of the invention is to provide with an easy, reliable and fast method to prepare in vitro, characterize and possibly recover immunogenic peptides. The invention is based on mixing in vitro at least one heat shock protein with peptides resulting from a in-vitro hydrolysis of at least one immunogenic and antigenic macromolecular structure.
Another aim of the invention is to provide with an easy, reliable and easily up-scalable method to prepare in-vitro and to characterize and possibly to recover a mixture of at least one heat shock protein with peptides resulting from the in-vitro hydrolysis of the said immunogenic and antigenic macromolecular structure.
Another aim of the invention is to provide with a method to separate and recover the peptides that do not bind to heat shock protein and the peptide-heat shock protein complexes, from a mixture according to the invention. Another aim of the invention is to characterize and possibly recover such unbound peptides.
Another aim of the invention is to provide with a method to isolate from the peptides-heat shock protein complexes according to the invention, the peptides that bind to the heat shock protein, to characterize and possibly to recover such peptides.
The present invention relates to a pharmaceutical or food composition comprising an adequate pharmaceutical or food vehicle, a stress protein (also known as xe2x80x9cheat shock proteinxe2x80x9d or HSP) and at least one of the epitopes (conformational or sequential epitope) of an immunogenic and antigenic macromolecular structure, said structure inducing graft rejection, an allergic reaction or an autoimmune reaction. Preferably, the pharmaceutical or food vehicle of the composition is adequate for mucosal (in particular oral) or cutaneous administration.
Advantageously, the stress protein and the epitope naturally form in vitro a complex naturally (i.e. without formation of a covalent bond), as described by Roamn et al., Febs (1994), Fouri et al., The Journal of Biological Chemistry, Volume 269 No. 48, pp. 30470-30478 (1994), Palleros et al., The Journal of Biological Chemistry, Volume 269 No. 48, pp. 13107-13114 (1994), Grageroov and Gottesman, Journal of Molecular Biology, No. 241, pp. 133-135 (1994), and Schmid et al., Science, Volume 260, p. 1991 (1994) incorporated below by way of reference.
Advantageously, the stress protein is a bacterial stress protein present, for example, in saprophytic bacteria, such as E. coli. 
Among the stress proteins of the present invention, mention may be made of the stress protein GroEL, the stress proteins GrpE, DnaK or DnaJ as described in particular by Hendrick and Hartl (Annual Review of Biochemistry, No. 62, p. 349 (1993)) or the heat shock proteins HSP 60, 70, etc.
The expression xe2x80x9cphenomenon of graft rejection or allergic or autoimmune reactionxe2x80x9d means hypersensitivity reactions of immediate or delayed type brought about by contact in particular with an allergen (this reaction can be immediate and specific (anaphylaxis, urticaria, etc.) or delayed over time) or autoimmune diseases and disorders of the immune system of immediate or delayed type associated with graft rejections of host against graft type and graft against host type.
Autoimmunity is a state of immunization of an individual against his or her own constituents, and the phenomenon of graft rejection is a state of immunization of an individual against foreign constituents (bodily fluids such as blood, cerebrospinal fluid, etc., cells, tissues, organs, antibodies, etc.) deliberately implanted into the patient. These phenomena are observed in particular in pathologies selected from the group consisting of infections associated with SLE (Systemic Lupus Erythematosus disease), Gougerot-Sjxc3x6gren syndrome (or Sjxc3x6gren""s disease) and rheumatoid polyarthritis, as well as pathologies such as sarcoidosis and osteopenia, spondylarthritis, scleroderma, multiple sclerosis, amyotrophic lateral sclerosis, hyperthyroidism, Addison""s disease, autoimmune hemolytic anemia, Crohn""s disease, Goddpasture""s syndrome, Graves"" disease, Hashimoto""s thyroiditis, idiopathic purpural hemorrhage, insulin-dependent diabetes, myasthenia, pemphigus vulgaris, pernicious anemia, poststreptococcal glomerulonephritis, psoriasis and spontaneous sterility, as well as immediate or delayed phenomena observed during graft rejections.
The expression xe2x80x9cimmunogenic and antigenic macromolecular structure which induces graft rejection or an allergic or autoimmune reactionxe2x80x9d means macromolecules such as allergens made of peptides, lipides, polysaccharides and/or nucleic acids, preferably selected from the group consisting of the major allergic antigens present in chemicals (latex), in foods such as eggs, soya and milk, in particular bovine beta-lactoglobulin (BLG) from cow""s milk, the major allergic antigens present in plants, molds, medicaments (in particular antibiotics or vaccines) and pollens, the major allergic antigens present in animals, in particular in hairs, and venom, in particular wasp and other insects venom, the major antigens of the allergic reaction to acari, to the mite present in house dust (antigen P1 Dermatophagoides pteronyssinus), the major antigen of Aspergillus fumagatus, and staphylococcal enterotoxin B (SEB).
Other non-limiting examples of allergens or mixtures of allergens have also been described in the publication ISBN-91-970475-5-4 by Pharmacia AB, which is incorporated herein by way of reference.
The xe2x80x9cmacromolecular structurexe2x80x9d can also be an antigenic complex made of peptides, liquids, saccharides and/or nucleic acids which induces an autoimmune disease. Preferably, this immunogenic and antigenic macromolecular structure is specific to lupus (SLE) or Sjxc3x6gren""s disease, in particular the plasma membrane or a portion of this membrane containing membrane DNA with a weight of greater than 100 KD, in particular as described in patent application WO 96/13723, the publication number of which is incorporated by way of reference.
Other non-limiting examples of antigenic complexes which induce autoimmune diseases have also been described by Roitt I. M. (Essential Immunology, Blackwell Scientific Publication (ch. 14) ISBN 0-632-01994-8) and by Humbel R. L. (Auto-anticorps et maladies auto-immunes [Autoantibodies and autoimmune diseases], Ed. Scientifiques Elsevier (1994) ISBN 2-906077-58-5).
This immunogenic and antigenic macromolecular structure can also be major histocompatibility loci (MHC I and/or MHC II) or minor histocomptability immunogenic and antigenic macromolecular structures present at the surface of blood cells and involved in the induction of the immune system (or portions thereof), which are specific to an individual and are involved in graft rejection phenomena (including bodily fluid transfusions).
The appropriate pharmaceutical or food vehicle according to the present invention may be any suitable additive or support, such as a nontoxic compatible substance for administration of the composition according to the invention to a patient. The type of appropriate pharmaceutical or food vehicle used will depend on the mode of administration selected. In particular, for oral administration, these vehicles can consist of aqueous solutions, syrups, lozenges, capsules, etc. Other pharmaceutical vehicles such as creams or ointments may be selected depending on the type of administration, in particular for cutaneous administrations.
A person skilled in the art can also adapt the pharmaceutical vehicle as a function of a subcutaneous, intradermal, intravenous, intramuscular or parenteral administration, via nasal or oral inhalation, etc.
The peptide and epitope according to the invention are advantageously (possibly) added to a specific emulsion composition, more preferably an oil in water emulsion which is suitable for specific mucosal administrations (especially an oral administration).
The percentage of active compound present in the composition according to the invention will depend on the type of patient, the pathology treated and the route of administration. The doses will be limited only by the patient""s tolerance to the product, as well as by the administration rates.
The administration concentrations will be chosen in particular such that the abovementioned pathological signs and symptoms are reduced, preferably eliminated, by the administration doses envisaged by the posology. The preferred concentrations for a human are: mg(active compounds)/Kg(patient).
The inventors have discovered, unexpectedly, that the use of the pharmaceutical and/or food composition according to the invention makes it possible to modify the immune response of a patient induced with said immunogenic and antigenic macromolecular structure. The modification of a patient""s immune response can be detected and quantified in particular according to the process and technique described in patent application WO 96/36880 or by any method of clinical analysis of the treated patient (including prophylactic methods) which is well known to those skilled in the art.
Another aspect of the present invention relates to the use of the composition according to the invention for the preparation of a medicament designed to modify a patient""s immune response toward an immunogenic and antigenic macromolecular structure which induces graft rejection or an allergic or autoimmune reaction. In particular, the present invention relates to the use of the pharmaceutical and/or food composition according to the invention for the preparation of a medicament intended to desensitize atopic or non-atopic allergies.
Another aspect of the present invention relates to the use of the pharmaceutical and/or food composition according to the invention for the preparation of a medicament intended for the prevention or treatment of the abovementioned allergic reactions and autoimmune diseases, for the treatment or prevention of graft rejections, optionally in combination with a specific product for reducing or neutralizing allergic reactions, autoimmune reactions and graft rejection phenomena (in particular the administration of immunosuppressants such as azathioprine, steroids, antilymphocyte globulins, cyclosporin A, rapamycin, FK-506 (tacrolimus) or lymphokines (in particular IL-10), and the analogs and agonists thereof which are well known to those skilled in the art.
The terms xe2x80x9canalogsxe2x80x9d and xe2x80x9cagonistsxe2x80x9d of these molecules means other molecules, or derivatives of these molecules, which act on the same receptor or via the same mechanism of action as the abovementioned specific products.
The present invention also relates to a process for the therapeutic or prophylactic treatment of a patient, comprising the step of administration of the composition according to the invention to said patient so as to modify the patient""s immune response toward an immunogenic and antigenic macromolecular structure which induces graft rejection or an allergic or autoimmune reaction.
The invention is related to any product comprising a mixture of at least one carrier molecule selected from the group consisting of heat shock proteins, antibodies, major locus histocompatibility complex (MHC) or similar molecules and peptides resulting from the in-vitro hydrolysis of at least one immunogenic protein.
The invention is related to any product comprising a mixture according to the invention of:
peptides resulting from said in-vitro hydrolysis of at least one immunogenic protein,
complexes between said carrier molecule and the peptides resulting from said in-vitro hydrolysis
uncomplexed carrier molecules.
The invention is also related to any product comprising purified complexes between at least said carrier molecule and peptides resulting from said in-vitro hydrolysis.
The invention is related to any product comprising the peptides isolated from the complexes between a said carrier molecule and peptides resulting from the said in-vitro hydrolysis.
The invention is also related to any pharmaceutical or food compositions comprising any product according to the invention. In a preferred embodiment, the products according to the invention are associated with immuno-modulators like but not limited to cytokines, anti-cytokines, corticoids, vaccine adjuvants, antibodies, thymic peptides, nanoparticles, emulsions and liposomes. In another embodiment, the composition comprise at least two carrier molecules complexes according to the invention.
The invention is also related to any pharmaceutical or food compositions according to the invention able to enhance an immune response against the entire protein through, but not limited to, T-cells cytotoxic immune response or to down-regulating immune response against the entire protein or any autoimmune diseases associated protein through, but not limited to, the mechanisms described by H. L. Wiener in Encyclopedia of Immunology, 2nd edition Academic Press (Ed P. J. DELVE and I. M. ROITT) pages 1893-1899 or by W. O Weigle in Encyclopedia of immunolgy 2nd edition Academic Press (Ed P. J. DELVE and I. M. ROITT) pages 2359-2361 or by D. W Scott in Encyclopedia of Immunology 2nd edition Academic Press (Ed P. J. DELVE and I. M. ROITT) pages 2362-2367. In a preferred embodiment the tolerance mechanism is a low dose mechanism. In another preferred embodiment the composition according to the invention induces TGF-xcex2 producing T-cells named Th3 T-cells.
The invention is also related to any diagnosis kit including any product according to the invention. In a preferred embodiment, the product according to the invention included in the diagnosis kit comprises at least two peptides-carrier molecule complexes according to the invention.
In a preferred embodiment of the invention, the proteins are:
any protein or other macromolecular structure or recombinant protein or mutated protein or combination thereof related to an autoimmune disease among other insulin, thyroglobulin, type II collagen, gliadin, GAD65, proteolipid protein, S-antigen, acetylcholin receptor, haptenized colonic proteins, interphotoreceptor retinoid binding protein, myelin, peripheral nerve P2, LDL, HDL
any protein or other macromolecular structure or recombinant protein or mutated protein or combination thereof related to allergy like the allergens according to Diagnostic Testing of Allergic disease Clinical Allergy and Immunology S. F. Kemp and R. F Lockey ISBN 0-8247-0303-0 pages 13-44.
any protein or other macromolecular structure or recombinant protein or mutated protein or combination thereof related to allograft and/or xenograft rejection like xenoantigen, alloantigen, MHC-peptides and blood group antigens.
any protein or other macromolecular recombinant protein or mutated protein or combination thereof from pathogens responsible of any infectious or parasitic diseases like virus, bacteria, fungi, protozoa and helminth.
any carrier molecule (heat shock protein related to autoimmune diseases or any recombinant protein or mutated protein or combination thereof or blood proteins).
In another preferred embodiment, the macromolecular structure, especially the protein, the recombinant protein, the mutated protein or the combinations thereof are extensively washed in an adequate solution through, but not limited to, ultra-filtration or dialysis to remove any low molecular weight material loosely associated with it.
Hydrolysis can be performed by enzymatic digestion with at least one protease or other suitable enzyme of any living organism. The proteases could be selected among the list according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology at http://www.chem.qmw.ac.uk/iumbmb/enzyme/EC34, and the list of the MEROPS database http://www.merops.co.uk and of the Rawlings N D and Barrel A J MEROPS: the peptidase database; Nucl. Acids Res. 28 323-325 (1998), and of Barret A J, Rawlings N D Woessner J F (eds) 1998 Handbook of Proteolytic Enzymes, Academic Press London.
In a preferred embodiment, macromolecular structures, especially proteins, known to be resistant to in-vitro or in-vivo hydrolysis are denatured either by physical (e.g. heating, high mechanical pressure) or by chemical methods (e. g. reductive reagents, urea, guanidinium chloride).
In another preferred embodiment, the structures resistant to hydrolysis are allergens (e.g. OVA).
In another preferred embodiment, the macromolecular structures with a proteolytic activity are denatured or specifically inhibited before enzymatic digestion (e. g; Der p1).
Hydrolysis can also be performed with at least one chemical agent like, but not limited to, mild acid (70% formic acid at 40xc2x0 C.), hydroxylamine, cyanogen bromide, iodosobenzoic acid or 2-nitro-5-thiocyanobenzoate followed by alkali.
In a preferred embodiment, the peptides or glycopeptides resulting from in-vitro hydrolysis of at least one macromolecular structure can be separated on the basis of their molecular weight e.g. by ultra-filtration or gel filtration or dialysis prior to the mixing with the heat shock proteins. In a preferred embodiment, the cut-off of the filter or of the membrane is about 10 kDa and at most 10 kDa (other suitable filters to be used have cut-offs of about 5 or 50 kDa).
In a preferred embodiment, the peptides or glycopeptides resulting from the in-vitro hydrolysis can be separated on the basis of their hydrophilic/hydrophobic properties e.g. by phase separation or solvent extraction before the mixing with the carrier molecule. Prior to the mixing with the carrier molecule, the organic phase is removed e.g. by solvent stripping or solvent evaporation or lyophilisation and hydrophobic peptides are dissolved in an adequate water solution e.g. a suitable buffer to make the complexes with the HSP.
In a preferred embodiment, the peptides or glycopeptides resulting from the in-vitro hydrolysis are bound to a solid support e.g. by covalent link, hydrophobic interactions, interactions between biotin or biotin derivatives and an avidin selected among the avidin protein families.
Characterization of the peptides or glycopeptides resulting from said hydrolysis can be performed either by ion exchange chromatography, or by gel electrophoresis, gel filtration or electro-elution, or by reverse phase high pressure liquid chromatography (HPLC).
Peptides that were bound to the HSP can be individually collected after HPLC or ion exchange chromatography elution, or by gel electrophoresis, gel filtration or electro-elution.
N-terminal peptides sequence analysis can be performed by procedures based upon the Edman degradation reaction. C-terminal peptides sequence analysis can be performed by using non specific carboxypeptidases (A, B, P, Y) . In another embodiment said peptide sequence can be performed by mass spectrometry analysis (e. g. MALDI or MALDI-TOF or ESI or LC-MS or MS/MS or FAB).
Peptides-carrier molecule complexes according to the invention can be characterized directly from the crude mixture by mass spectrometry e.g. MALDI or MALDI-TOF.
Carrier molecules, especially the heat shock proteins according to the invention are molecular chaperones according to Mary-Jane Gething xe2x80x9cGuidebook to molecular chaperones and protein folding catalystsxe2x80x9d A Sambrook and Tooze publication at Oxford University Press and xe2x80x9cMolecular Chaperones and Folding Catalystsxe2x80x94Regulation, Cellular Function and Mechanismsxe2x80x9d B. Bukauxe2x80x94Harwood Academic publishers. In a preferred embodiment of the invention, the heat shock proteins are purified and depleted of any bound-peptides according to the method known by the person skilled in the art, e.g. incubation in a low-pH buffer or in an ATP-containing buffer.
In another preferred embodiment, the heat shock proteins are partial or total recombinant heat shock proteins or any homologous sequences of said HSP described previously. In another preferred embodiment the heat shock protein are microbial heat shock protein (bacteria, fungi and yeast).
In a preferred embodiment, the carrier molecules are bound to a solid support, e.g. by covalent links, hydrophobic reactions, interactions between biotin and biotin derivatives and an avidin chosen among the avidin protein families, gelatin, ATP- or ADP affinity column.
The mixture is obtained by mixing in vitro at least one solution of peptides resulting from an in vitro hydrolysis of at least one protein with at least one solution containing at least one carrier molecule. In this case it is bound to a solid support, at least one solution of peptides resulting from the hydrolysis of at least one carrier molecule is put into contact with the solid support. In the case of peptides resulting from the in-vitro hydrolysis of at least one protein are bound to a solid support, at least one solution of at least one carrier molecule is put into contact with the solid support. In a preferred embodiment, the mixing is performed under conditions allowing the formation of peptide-carrier molecule complexes. Such conditions are described in the prior art.
The compositions according to the invention are prepared as a galenic form to modulate the immune response.